1. Field of the Invention
This invention is referred to a new bacterial strain of Bacillus sp., useful to be applied in a metal biosorption process. It is described, besides, a sporulated and non-sporulated industrial inoculant of said bacterium, a method to produce said inoculant and a process to remove metals by said bacterium.
2. Background
Biosorption is understood as the uptake of heavy metals by biomass (living or nonliving) by physical-chemical mechanisms purely. In general terms, the biosorption process has been described as a non-selective mechanism that allows the removal of the following metals: Ag, Al, Au, Co, Cd, Cu, Cr, Fe, Hg, Mn, Ni, Pb, Pd, Pt, U, Th, Zn, and others (Gadd and White (1993). Microbial Treatment of Metal Pollution—A Working Biotechnology? Trends Biotechnol., 11, 353-359).
Nowadays, there is a large amount of international scientific literature about the capability of some microorganisms of concentrating metal ions by biosorption from diluted solutions, among them we can mention Castro et al. (1992), Biomasa de Rhizopus oligosporus como adsorbente de iones metálicos. Microbiología SEM 8, 94-105, Cotoras et al. (1992), Biosorption of metal ions by Azotobacter vinelandii. World Journal of Applied Microbiology and Biotechnology 8, 319-323, Cotoras et al. (1992), Sorption of metal ions by whole cells of Bacillus and Micrococcus Environmental Technology 13, 551-559, Fourest and Roux (1992), Heavy metal biosorption by fungal mycelial by-products: mechanism and influence of pH. Appl. Microbiol. Biotechnol. 37 pp. 399-403, Holan and Volesky (1995), Accumulation of cadmium, lead and nickel by fungal and wood biosorbents. Appl. Biochem. Biotechnol. 53 pp. 133-142. The research works have covered different groups of organisms, among which are: bacteria (e.g. E. coli, Zoogloea ramigera, Bacillus subtilis, Azotobacter vinelandii, etc.), fungi (e.g. Rhizopus arrhizus, Aspergillus niger), and algae (e.g. Chlorella vulgaris, Sargassum sp.). There are many reviews on the state of the art of the application of biosorption and its advances. Among them, are: Kratochvil D. and B. Volesky, Advances in the biosorption of heavy metals, Trends Biotechnol. 16 (1998), pp. 291-300; Volesky B. and Z. R. Holan, Biosorption of heavy metals, Biotechnol. Prog. 11 (1995), pp. 235-250; Kapoor, A. and Viraraghavan, T. (1995), Fungal Biosorption—An Alternative Treatment Option for Heavy Metal Bearing Wastewaters: A Review. Bioresource Technology, 53, 195-206 and Volesky Detoxification of metal-bearing effluents: biosorption for the next century Hydrometallurgy Volume 59, 203-216 (2001). From all of these researches it is possible to conclude that microorganisms can concentrate important amounts of metal ions. Values of biosorption from 0.3% to 35% of the microbial dry weight when using solutions with metals concentration between 10 and 100 mg/L have been described.
On the other hand, it is important to consider that a series of strains of the Bacillus type involved in processes of recovering of different metals have been described. An example: U.S. Pat. No. 5,005,130, which describes a method to recover silver from a refractory mineral of manganese dioxide and silver by using manganese-reducing bacterium Bacillus polymyxa and, particularly, Bacillus polymyxa, strain D-1, ATCC-55030, the method is based on solubilizing manganese, concentrating the silver. In the same direction, U.S. Pat. No. 5,422,268 introduces a process to recover plutonium from plutonium-polluted soils: manganese-reducing bacterium Bacillus circulans SD-1 NRRL B-21037 to release plutonium from soils by solubilization, and patent WO9214848, shows the application of manganese-reducing bacterium Bacillus sp. MBX 69 NRRL B-18768 to recover different metals from a mineral of manganese dioxide or polluted soils.
One of the most relevant requirements for the technological application of biosorption is the biomass retention, in order to allow the biosorbent to be kept in a reactor, so it can be reused. This has been frequently performed by immobilizing the microorganisms in a matrix. There are many examples on the application of these methodologies, such as the outstanding works by Brierley (Brierley, Production and application of a Bacillus-based product for use in metals biosorption. In: B. Volesky, Editor, Biosorption of Heavy Metals, CRC Press, Boca Raton, Fla. (1990), pp. 305-312; Brierley and Brierley, Immobilization of biomass for industrial application of biosorption. In: A. E. Torma, M. L. Apel and C. L. Brierley, Editors, Biohydrometallurgical Technologies, Proceedings of the International Biohydrometallurgy Symposium, The Minerals, Metals and Materials Society, Warrendale, Pa. (1993), pp. 35-44), who created an immobilized biosorbent based on a bacterium (Bacillus subtilis).
Volesky, et al., (1988) patented a method for gold biosorption using the biomass of brown seaweed attached by a natural or synthetic polymer (U.S. Pat. No. 4,769,223). In the 90's decade, the greatest part of the patents followed this example, protecting the production of pellets-shaped biosorbents by means of the artificial winning or immobilization of the biomass. This focus is also used by the following patents: Brierley, et al. (1990, U.S. Pat. No. 4,898,827) they use immobilized Bacillus subtilis with the metal attachment capacity of this bacterium, Greene, et al. (1991, U.S. Pat. No. 5,055,402) they used immobilized microalgae at high levels of temperature (300° C. to 500° C.). It is also important to mention the development of polymer beads, such as polysulfone to immobilize sorbents (Jeffers, et al., 1994, U.S. Pat. No. 5,279,745), which constitutes the base for BIO-FIX, developed by the Bureau of Mines of the United States. More recently, the following processes of preparation of biosorbents have been published: crosslinked yeasts by aldehydes (Yannai, et al. 1996, U.S. Pat. No. 5,538,645), biological materials beads immobilized by neutralized and crosslinked poli-(acid carboxylics) adhesive (Summers, Jr., et al. 1997, U.S. Pat. No. 5,602,071), brown seaweed which alginate has been extracted (Pohl 1997 U.S. Pat. No. 5,648,313), fungal biomass (of the types of Aspergillus, Penicillium y Trichoderma) or bacterial (Micrococcus) treated with phosphoric acid, solvents and sodium hydroxide (Kogtev, et al. 1998, U.S. Pat. No. 5,789,204), microorganisms immobilized in hydrophilic polyurethane (Hermann 1999, U.S. Pat. No. 5,976,847). More recently, Nakao and Suzuky (2004) introduced a metals-adsorbent composition containing Bacillus sp. KRI-02, Bacillus licheniformis, and Staphylococcus sp. KRI-04 bacterial cells, which is obtained by acid treatment of the bacteria (CA Patent 2497264).
Although these biosorbent materials are promising, they present the disadvantages of requiring a biomass concentration stage, usually by centrifugation, and the need of employing chemical agents that allow its immobilization as granulated or agglomerated products. Both are very expensive processes that demand a high energetic cost and employ toxic chemical products or environmental contaminants.
As an alternative, CL Patent 40704 (Cotoras and Viedma, 2000) and the patent application No 1945-2005 (Cotoras et al., 2005) describes a process in which, first of all, a biofilm is formed spontaneously on a low-priced-inert-support material. Once the immobilization is finished, the alternated cycles of biosorption and desorption start. Nevertheless, the available microorganisms in the state of the art (e.g. Pseudomonas, Klebsiella, Bacillus, etc.) show a series of disadvantages to achieve an efficient performance of this process. The main difficulties are: the inoculant must be produced and transported to the decontamination plant. This is limited by the low stability of the inoculant culture and the enormous volume required. In addition, the available strains existing nowadays have a low biofilm formation and contaminants removal capacity.
This invention presents a series of alternatives to the disadvantages of the technologies available in the state of the technique, because by isolating a bacteria strain that shows a high attaching capacity it is possible to form aggregates of vegetative cells or spores and form biofilms on a solid support material. The cell aggregates generation allows producing, in a simple way, a concentrated inoculant of bacterial cells, both vegetative and sporulated. On the other hand, the high biofilm formation capacity facilitates the colonization of the inert support material, increasing the contaminants removal capacity of the biosorption process.